ASSAY METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS QUANTITATIVE ESTIMATION OF DILOXANIDE FUROATE AND ORNIDAZOLE IN TABLETS BY RP-HPLC Original Article D. VIJAY KUMAR 1 , P. SWETHA 1 , G. SAI PRASAD 2 , A. ASHOK KUMAR 1 * 1 Department of Pharmaceutical Analysis and Quality Assurance, Vijaya College of Pharmacy, Munaganur (village), Hayathnagar (Mandal), Hyderabad 501511, India, 2 Received: 02 Feb 2015 Revised and Accepted: 02 Sep 2015 Chandra Labs, 5-5-35/173, Plot No-10, 1st Floor, IDA Prasanthi Nagar, Kukatpally, Hyderabad, India 500090 Email: ashok576@gmail.com ABSTRACT Objective: To develop an accurate, precise and linear Reverse Phase High Performance Liquid Chromatographic (RP-HPLC) method for simultaneous quantitative estimation of Diloxanide furoate and Ornidazole in tablets and validate as per ICH guidelines. Methods: The optimized method uses a reverse phase column, Waters Symmetry C18 (250 X 4.6 mm; 5μ), a mobile phase of tri ethyl ammonium phosphate buffer (pH 2.3):acetonitrile in the proportion of 40:60 v/v, flow rate of 1.0 ml/min and a detection wavelength of 270 nm using a UV detector. Results: The developed method resulted in Diloxanide furoate eluting at 4.31 min and Ornidazole at 2.64 min. Diloxanide furoate exhibited linearity in the range 46.87-140.62μg/ml, while Ornidazole exhibited linearity in the range 31.25-93.75μg/ml. The precision is exemplified by relative standard deviations of 1.02% for Diloxanide furoate and 0.514% for Ornidazole. Percentage Mean recoveries were found to be in the range of 98-102, during accuracy studies. The limit of detection (LOD) for Diloxanide furoate and Ornidazole were found to be 122.54µg/ml and 81.54µg/ml respectively, while limit of quantitiation (LOQ) for Diloxanide furoate and Ornidazole were found to be 371.33µg/ml and 247.11µg/ml respectively. Conclusion: A simple, accurate, precise, linear and rapid RP-HPLC method was developed for simultaneous quantitative estimation of Diloxanide furoate and Ornidazole in tablets and validated as per ICH guidelines. Hence it can be used for the routine analysis of Diloxanide furoate and Ornidazole in tablets in various pharmaceutical industries. Keywords: RP-HPLC, Diloxanide furoate, Ornidazole, Method development, Validation. Ornidazole (fig. 1) chemically is 1-(3-chloro-2-hydroxypropyl)-2- methyl-5-nitroimidazole. It has a molecular formula of C INTRODUCTION 7 H10ClN3O3 and a molecular weight of 219.625 g/mol. Ornidazole is a derivative of 5-nitro imidazole used as an anti-infective agent [1]. Ornidazole is converted into an active form by reduction of its nitro group to amine that binds to microbial DNA and prevents nucleic acid formation, belonging to class of bacteriostatic [2]. Ornidazole is used for the treatment of bacterial vaginosis, trichomoniasis, genitourinary infections in women and men, amoebiasis, giardiasis. It is also used in infections against anaerobic bacteria and in the treatment of prophylaxis during surgical interventions, particularly those involving the colon, and in gynaecological operations [2]. Ornidazole has been successfully employed in combination with other drugs for peptic ulcers, few types of gastritis, stomach cancers, rheumatoid arthritis [3] and in the prophylaxis of Crohn’s disease [4]. N N CH 3 Cl OH O 2 N Fig. 1: Structure of Ornidazole Diloxanide furoate (fig. 2) chemically is 4-(N-methyl-2,2- dichloroacetamido) phenyl-2-furoate having the molecular formula as C 14H11 Cl 2NO4 and the molecular weight as 328.147 g/mol [5]. It is an effective drug for the treatment of asymptotic persons who are passing cysts of Entameba histolytica [6]. It acts principally in the bowel lumen and is used in the treatment of the intestinal amoebiasis. Diloxanide furoate has been used in the treatment of the asymptotic carriers of Entameba histolytica [6] and is excellent amoebicide for cyst passers [7, 8]. O N Cl Cl C H 3 O O O Fig. 2: Structure of Diloxanide furoate A detailed literature survey reveals that there exists literature on chromatographic methods for Ornidazole alone and in combination with other drugs [9-16] and similarly Diloxanide furoate in combination with other drugs [17-22] in various matrices. While there is only one literature on RP-HPLC assay methods for the simultaneous quantitative estimation of Diloxanide furoate and Ornidazole in pharmaceutical dosage forms using mixed phosphate buffer at pH 6.0[23]. As there is no literature reported using triethylammonium phosphate buffer as aqueous media along with acetonitrile as mobile phase, we here report a new and a rapid RP- HPLC validated method for the simultaneous quantitative estimation of Diloxanide furoate and Ornidazole in tablets using triethylammonium phosphate buffer (pH 2.3) as per ICH guidelines. MATERIALS AND METHODS Chemicals and reagents Analytically pure sample of Diloxanide furoate and Ornidazole with purities greater than 95% were obtained as gift samples from International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 7, Issue 10, 2015 Innovare Academic Sciences